Transglutaminases (TGs) are highly regulated, calcium-dependent enzymes that likely play key roles in several critical processes in the nervous system. TGs catalyze a transamidating reaction that results in the incorporation of polyamines into specific glutatmine residues within proteins, or if a protein-bound lysine is the amine donor, the tTG-catalyzed reaction results in the formation of a crosslink between the glutamine and lysine residues in proteins. This is highly specific reaction and only a limited number of proteins have been identified as in situ substrates of TG. Tissue TG (tTG), a member of this family, is the most abundant TG in the human brain and is found within neurons. The levels of tTG and TG activity are increased in neurodegenerative conditions such as Alzheimer's disease and Huntington's disease, and increases in tTG can facilitate neuronal cell death in response to specific stressors. Further, in a recent study it was found that treatment with cystamine, a drug that inhibits transglutaminase in vitro, prolonged surviv approximately 8-10% in a mouse model of Huntington's disease. These and other results suggest that transglutaminase inhibitors may be beneficial in studying the pathological mechanisms and in the treatment of neurodegenerative diseases such as Huntington's Disease and Alzheimer's Disease. However, there are numerous problems with using cystamine as a drug to treat human neurodegenerative diseases, including the fact that it is a non-specific drug with a very steep toxicity curve. Further, it is a charged molecule that likely does not efficiently cross the blood brain barrier. Therefore it is clear that more selective and efficacious TG inhibitors need to be developed for the study and treatment of neurodegenerative disorders. This laboratory has a longstanding interest in the regulation and function of TGs, and therefore is optimally suited to develop TG assays, both in vitro and in situ, that can be used in high throughput drug screening to identify and develop TG inhibitors that are neuroprotective. The aims of this proposal are to: 1. To develop an assay to measure TG activity for a 96 well format that is robust, reproducible and has a simple readout. 2. To develop an assay to measure TG activity in a cellular model using a 96 well format that is reproducible and easily measured. 3. To validate the assays using pharmacological standards, and then use these assays in an initial screen of a small but diverse collection of FDA approved drugs to demonstrate feasibility.